Polyhydroxyalkanoic acids are polyester-type organic polymers produced by a wide range of microorganisms. These polymers are biodegradable, are thermoplastic polymers, and are producible from recyclable resources, so that attempts for an industrial production of the polymers as environment-conscious materials or biocompatible materials to be used for various industries have been conducted.
The monomer constituting said polyester is 3-hydroxyalkanoic acid in the general name. Specifically, its polymer molecule is formed by homopolymerization or copolymerization of 3-hydroxybutyric acid, 3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, or 3-hydroxyalkanoic acid having a longer alkyl chain. Poly 3-hydroxybutyric acid (hereinafter referred to briefly as “P(3HB)”), which is a homopolymer of 3-hydroxybutyric acid (hereinafter referred to briefly as “3HB”), was first discovered in Bacillus megaterium in 1925. However, since P(3HB) is high in crystallinity, it is hard and brittle, so that the range of practical application thereof is limited. Therefore, studies have been undertaken to improve these properties.
Among others, a process for producing a copolymer made of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) (hereinafter such copolymer is referred to briefly as P(3HB-co-3HV)) is disclosed (for example, refer to Japanese Kokai Publication Sho-57-150393 and Japanese Kokai Publication Sho-59-220192). This P(3HB-co-3HV) is rich in flexibility as compared with P(3HB), hence was expected to have a broader application range. In actuality, however, P(3HB-co-3HV) shows only slight changes in physical properties even when the molar fraction of 3HV is increased. In particular, the flexibility can not be improved. Thus, it has been used only in the field of rigid shaped articles such as shampoo bottles and disposable razor grips.
Moreover, a medium-chain PHA constituted of 3-hydroxyalkanoic acids having 6 to 16 carbon atoms is known to be in lower crystallinity as compared with P(3HB) or P(3HB-co-3HV), and rich in elasticity (refer to Madison et al., Microbiol. Mol. Biol. Rev., 63:21-53 (1999)). Thus, applications for different fields are hoped for. Studies for producing the medium-chain PHA has been conducted by introducing a PHA synthase gene of the genus Pseudomonas into the genus Pseudomonas, the genus Ralstonia, or Escherichia coli, but all of these processes were not suitable for the industrial production due to low productivity (refer to Matsusaki et al., J. Bacteriol., 180:6459-6467 (1998); Matsusaki et al., Appl. Micrbiol. Biotechnol., 53:401-409 (2000); and Langenbach et al., FEMS Microbiol. Lett., 150:303-309 (1997)).
In recent years, studies have been made concerning the copolymer consisting of two components 3HB and 3-hydroxyhexanoic acid (hereinafter referred to as 3HH for short) (hereinafter such copolyesters are referred to as P(3HB-co-3HH) for short) and the process for producing it (refer to Japanese Kokai Publication Hei-05-93049 and Japanese Kokai Publication Hei-07-265065). According to these patent documents, this process for producing P(3HB-co-3HH) comprises fermentative production thereof from fatty acids, such as oleic acid, or oils and fats, such as olive oil, using Aeromonas caviae isolated from soil. Studies concerning the properties of P(3HB-co-3HH) have also been made (refer to Doi et al., Macromolecules, 28:4822-4828 (1995)). According to this report, when Aeromonas caviae is cultured using fatty acids of not less than 12 carbon atoms as the only carbon source, P(3HB-co-3HH) with a 3HH composition of 11 to 19 mole percent can be fermentatively produced. It has been revealed that the properties of such P(3HB-co-3HH) change from hard and brittle gradually to soft and flexible, to an extent exceeding the flexibility of P(3HB-co-3HV), with the increase in the 3HH composition. That is, P(3HB-co-3HH) can be given a wide applicable range of physical properties, from properties of rigid polyesters to properties of flexible polyesters, by changing the 3HH composition and therefore can be expected to be applicable in a wide range, from a chassis for a TV-set and the like, for which rigidity is required, to films and the like, for which flexibility is required. However, with this production process, the productivity of polyester was as low as the cell production of 4 g/L and polyester content of 30%, and the process has yet to be said insufficient as the process for the practical use of the polyester. Therefore, a process providing higher productivity has been searched for toward the practical use.
Attempts aiming at the industrial production of P(3HB-co-3HH) have also been conducted. Among cultures using Aeromonas hydrophila, in a 43-hour feed culture using oleic acid as a carbon source, P(3HB-co-3HH) with the cell productivity of 95.7 g/L, polyester content of 45.2% and 3HH composition of 17% was produced (refer to Lee et al., Biotechnol. Bioeng., 67:240-244 (2000)). Furthermore, Aeromonas hydrophila was cultured using glucose and lauric acid as carbon sources, and the cell productivity of 50 g/L and polyester content of 50% were attained (refer to Chen et al., Appl. Microbiol. Biotechnol., 57:50-55 (2001)). However, Aeromonas hydrophila has pathogenicity to human (refer to “Safety Control Regulations on Pathogen, etc.” issued by National Institute of Infectious Diseases; attached table 1, appended chart 1 (1999)), and thus cannot be said as a suitable species for the industrial production. Moreover, since expensive carbon sources are used in these culture productions, use of a cheap carbon source has also been asked for in view of the production cost.
Therefore, attempts for the production using a safe host and improvement of the productivity have been conducted. A polyhydroxyalkanoic acid (PHA) synthase gene was cloned from Aeromonas caviae (refer to Japanese Kokai Publication Hei-10-108682; and Fukui et al., J. Bacteriol., 179:4821-4830 (1997)). As a result of producing P(3HB-co-3HH) using a transformant prepared by introducing this gene into Ralstonia eutropha (formerly Alcaligenes eutrophus), the cell productivity was 4 g/L and the polyester content was 30%. Moreover, as a result of culturing this transformant using a vegetable oil as a carbon source, the cell productivity of 4 g/L and polyester content of 80% were attained (refer to Fukui et al., Appl. Microbiol. Biotecnol., 49:333-336 (1998)). Furthermore, researches on culture processes have also been conducted as can be seen in improvements of the cell productivity, polyester content and 3HH composition of up to 45 g/L, 62.5%, and 8.1%, respectively, by improving culture conditions (refer to Japanese Kokai Publication No. 2001-340078).
Moreover, Ralstonia eutropha capable of producing P(3HB-co-3HH) using fructose as a carbon source was also constructed, but this strain had low polyester productivity, and cannot be said to be suitable for the practical production (refer to Fukui et al., Biomacromolecules, 3:618-624 (2002)).
A P(3HB-co-3HH)-producing strain using Escherichia coli as the host was also constructed. A strain prepared by introducing a PHA synthase gene of the genus Aeromonas, NADP-acetoacetyl Co-A reductase gene of Ralstonia eutropha, or the like, into Escherichia coli was constructed. As a result of culturing said Escherichia coli using dodecane as a carbon source, the cell productivity of 79 g/L, polyester content of 27.2%, and 3HH composition of 10.8% were obtained (refer to Park et al., Biomacromolecules, 2:248-254 (2001)).
With the aim of improving the productivity of P(3HB-co-3HH) and controlling the 3HH composition, artificial modification of PHA synthase was carried out. Among PHA synthase mutants derived from Aeromonas caviae, a mutant enzyme in which serine is substituted for 149th amino acid asparagine and a mutant enzyme in which glycine is substituted for 171st aspartic acid showed improved PHA synthase activity in Escherichia coli and 3HH composition (refer to Kichise et al., Appl. Environ. Microbiol., 68:2411-2419 (2002)). Moreover, it was reported that a mutant enzyme in which isoleucine is substituted for 518th phenylalanine or a mutant enzyme in which glycine is substituted for 214th valine were improved in the PHA synthase activity in Escherichia coli and polyester content (refer to Amara et al., Appl. Microbiol. Biotechnol., 59:477-482 (2002)). However, since these mutants used particular Escherichia coli species as the host and still showed low polyester content, further improvements aiming at the industrial production making use of characteristics of these mutant enzymes have been required.
One of the most important subjects when PHAs are cultured in the industrial scale using a strain produced by applying recombinant DNA technologies is the stability of the transgene. For the gene transfer, a process comprising using a plasmid, a process comprising incorporation onto a host chromosome, and the like are used. However, a plasmid is known to be lost during proliferation and division of recombinant cells (refer to Japanese Kokai Publication Sho-59-205983). Therefore, cells from which a plasmid is lost lose PHA production ability, which leads to decrease of the commercial productivity. Conventionally, recombinant cells are generally cultured by selectively growing and retaining plasmid retention cells alone by adding antibiotics to a medium. However, problems such as a cost increase by the use of antibiotics, or influences to environment by residual antibiotics in a waste culture solution occur. Moreover, for stabilizing the plasmid, recombinant Escherichia coli prepared by introducing parB gene derived from R1 plasmid into P(3HB)-producing plasmid was produced (refer to Lee et al., J. Biotechnol., 32:203-211 (1994)). This Escherichia coli retained almost 100% of the plasmid even after culturing 110 to 120 generations. However, the plasmid still has the risk of being lost.
On the other hand, a gene incorporated onto a chromosome is thought to be stable. For this reason, a microorganism in which a gene involved with PHA synthesis was incorporated onto the chromosome was reported (refer to U.S. Pat. No. 6,593,116, Kranz et al., Appl. Environ. Microbiol., 63:3003-3009 (1997); and York et al., J. Bacteriol., 183:4217-4226 (2001)).
The Escherichia coli strain produced by randomly inserting a gene coding for a PHA biosynthesis enzyme onto the chromosome of Escherichia coli, as disclosed in U.S. Pat. No. 6,593,116, produced P(3HB) at the level exceeding 85% of the dry cell weight. However, for making Escherichia coli produce P(3HB-co-3HH) having superior practical physical properties, it is required to make genes for supplying a substrate monomer coexist, or to supply expensive fatty acids, etc., to a medium. These requirements prevent attaining higher production efficiency. Furthermore, when the gene is randomly incorporated onto the chromosome, depending on the site to be incorporated, expression of the gene on that site or on the peripheral site thereof may be affected, and sufficient performances as a PHA-producing strain cannot be exhibited.
In the case of introducing an exogenous PHA synthase gene into a host in the form of plasmid or incorporating the same onto the chromosome, when a microbial species which inherently produces a PHA, such as the genus Ralstonia or the genus Pseudomonas, is used as the host, the strain which lost the inherent ability to produce a PHA has been used. Since such a strain is produced through mutation operations, the proliferation ability or biological activity is inferior to that of the parental strain (refer to Schlegel et al., Arch. Mikrobiol., 71:283-294 (1970); Schubert et al., J. Bacteriol., 170:5837-5847 (1988); Peoples et al., J. Biol. Chem., 264:15298-15303 (1989); and the like), and it is hard to say that such a strain exhibits sufficient production ability as the PHA-producing strain.
On the other hand, in the report in which a PHA synthase gene derived from Chromatium vinosum is substituted for a PHA synthase gene on the chromosome of Ralstonia eutropha, P(3HB) was accumulated at 91% to the dry cell weight, which exceeded wild strains (York et al., J. Bacteriol., 183:4217-4226 (2001)). However, the cell productivity was as low as 1.8 g/L, and also the produced polyester was hard and brittle P(3HB), not P(3HB-co-3HH) with which a wide range of applications can be expected. Thus, for the commercial production of the polyester, there still have remained subjects to be solved.